Prenylflavonoids isolated from Macaranga tanarius stimulate odontoblast differentiation of human dental pulp stem cells and tooth root formation via the mitogen-activated protein kinase and protein kinase B pathways.

AIM: To identify odontogenesis-promoting compounds and examine the molecular mechanism underlying enhanced odontoblast differentiation and tooth formation. METHODOLOGY: Five different nymphaeols, nymphaeol B (NB), isonymphaeol B (INB), nymphaeol A (NA), 3'-geranyl-naringenin (GN) and nymphaeol C (NC) were isolated from the fruit of Macaranga tanarius. The cytotoxic effect of nymphaeols on human DPSCs was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of nymphaeols on odontoblast differentiation was analysed with Alizarin Red S staining and odontoblast marker expression was assessed using real-time polymerase chain reaction and Western blot analysis. The molecular mechanism was investigated with Western blot analysis. In order to examine the effect of INB on dentine formation in the developing tooth germ, INB-soaked beads were placed under the tooth bud explants in the collagen gel; thereafter, the tooth bud explant-bead complexes were implanted into the sub-renal capsules for 3 weeks. Tooth root formation was analysed using micro-computed tomography and histological analysis. Data are presented as mean ± standard error (SEM) values of three independent experiments, and results are compared using a two-tailed Student's t-test. The data were considered to have statistical significance when the P-value was less than 0.05. RESULTS: Three of the compounds, NB, INB, and GN, did not exert a cytotoxic effect on human DPSCs. However, INB was most effective in promoting the deposition of calcium minerals in vitro (P < 0.001) and induced the expression of odontogenic marker genes (P < 0.05). Moreover, this compound strongly induced the phosphorylation of mitogen-activated protein (MAP) kinases and protein kinase B (AKT) (P < 0.05). The inhibition of p38 MAP, c-Jun N-terminal kinase (JNK), and AKT substantially suppressed the INB-induced odontoblast differentiation (P < 0.001). In addition, isonymphaeol B significantly induced the formation of dentine and elongation of the tooth root in vivo (P < 0.05). CONCLUSIONS: Prenylflavonoids, including INB, exerted stimulatory effects on odontoblast differentiation and tooth root and dentine formation via the MAP kinase and AKT signalling pathways. These results suggest that nymphaeols could stimulate the repair processes for dentine defects or injuries.

Incidence of venous thromboembolism following major surgery in Korea: from the Health Insurance Review and Assessment Service database.

BACKGROUND: Data on the incidence of venous thromboembolism (VTE) following major surgery in Asian populations are limited. METHODS: Using the Korean Health Insurance Review and Assessment Service database, we performed a nationwide population-based epidemiologic study to estimate the incidence of VTE after major orthopedic, cancer, and benign surgeries. VTE cases were identified from all patients undergoing major surgery between 2007 and 2011 using both diagnostic and drug codes as treatment evidence of VTE within 5 weeks of surgery. We also calculated the relative risk of VTE in major orthopedic and cancer surgery compared to benign surgery. RESULTS: The overall rates of postoperative VTE were 1.24%, 0.67%, and 0.05% for major orthopedic, cancer, and benign surgeries, respectively. Hip fracture (1.60%) and colorectal cancer surgeries (1.67%) were associated with the highest rates of VTE, and the rates steadily increased during the study period. Advanced age, female sex, and general anesthesia were independent risk factors for VTE. Patients undergoing surgery for colorectal, pancreatic, ovarian, and esophageal cancer, and major orthopedic surgery had a > 20-fold higher risk of VTE than those undergoing benign surgery. CONCLUSIONS: This is the largest epidemiologic study to investigate the incidence of VTE after major surgery in Asia, demonstrating that the rates of postoperative VTE are lower than in Caucasian populations. This study contributes to a better understanding of the differences in postoperative VTE development between Korean and Caucasian populations; the data also suggest that perioperative prophylactic strategies in Asians should be based on studies of such populations.

Sarcomatoid renal cell carcinoma in a renal transplant recipient.

The incidence of transplanted kidneys derived from elderly donors is increasing because of the larger waiting lists and greater age of patients with end-stage renal failure. Compared with young donors, one of the problems is the heightened risk of neoplasm transmission. We report 2 cases of kidney recipients, both of whom developed a sarcomatoid renal cell carcinoma after receiving a kidney transplant from the same 68-year-old male donor, who did not show signs of a neoplasm on a previous abdominal ultrasound or a pretransplant biopsy. The first recipient was a 66-year-old woman who developed a kidney mass with several urologic obstructive complications, tumor dissemination, and death at 9 months after kidney transplantation. The second recipient was a 48-year-old asymptomatic man with normal renal function, who was studied after the results of the first recipient, revealing another renal tumor. Transplant nephrectomy was performed and a peritoneal implant was resected. The patient is alive without evidence of a neoplasm after 18 months. Herein we have discussed the mechanisms of neoplasm transmission in kidney transplantation and possible strategies for its prevention and treatment.

The effect of pneumoperitoneum in the steep Trendelenburg position on cerebral oxygenation.

BACKGROUND: daVinci robot-assisted laparoscopic radical prostatectomy (RALP) requires pneumoperitoneum in the steep Trendelenburg position, which results in increased intracranial pressure and cerebral blood flow. The aim of this study was to evaluate the effect of pneumoperitoneum in a 30 degrees Trendelenburg position on cerebral oxygenation using regional cerebral oxygen saturation (rSO2). METHODS: Thirty-two male patients of ASA I and II physical status without previous episodes of cerebral ischemia or hemorrhage undergoing daVinci RALP were enrolled. The rSO2 was continuously monitored with near-infrared spectroscopy (INVOS) 5100) during the study period. Measurements were obtained immediately after anesthesia induction (T0; baseline), 5 min after a 30 degrees Trendelenburg position (T1), 5 min after 15 mmHg pneumoperitoneum in a supine position (T2), 30, 60 and 120 min after the pneumoperitoneum in a Trendelenburg position (T3, T4 and T5, respectively) and after desufflation in a supine position (T6). RESULTS: The change in the left and right rSO2 was statistically significant (Left P=0.004 and Right P=0.023). Both the right and the left rSO2 increased significantly during pneumoperitoneum in a Trendelenburg position (from T3 to T5) and at T6 compared with the baseline value at T0. The partial pressure of carbon dioxide (PaCO2) was increased significantly at T2, T3, T5 and T6 compared with the baseline value at T0. CONCLUSIONS: During daVinci RALP, cerebral oxygenation, as assessed by rSO2, increased slightly, which suggests that the procedure did not induce cerebral ischemia. The PaCO2 should be maintained within the normal limit during pneumoperitoneum in a Trendelenburg position in patients undergoing daVinci RALP because the rSO2 increased in conjunctions with the increase in PaCO2.

A prospective randomized study on the mobilization of CD34+ cells comparing continuous intravenous vs subcutaneous administration of rhG-CSF in normal donors.

The efficacy of mobilizing peripheral blood progenitor cells (PBPC) with continuous intravenous (c.i.v.) administration of rhG-CSF was randomly compared to subcutaneous (s.c.) administration, in 15 normal donors in each arm of the study for 6 days. The percentage and absolute numbers of CD34+ cells in the c.i.v. and s.c. groups increased maximally at day 3 and 5, respectively, when compared with the steady-state (day 0) level. Peak CD34+ cell levels were achieved on day 3 in the c.i.v. group, with more rapid results than in the s.c. group (49.3/microl vs 35.9/microl, P=0.043). Plasma rhG-CSF levels declined progressively during mobilization in each group as the WBC increased. The serum level of rhG-CSF did not correlate with CD34+ cell counts in the peripheral blood. Toxicity profiles in the c.i.v. and s.c. groups were similar. Each regimen was effective in successfully mobilizing the target CD34 cell number.

Bran extracts from pigmented rice seeds inhibit tumor promotion in lymphoblastoid B cells by phorbol ester.

Using flow cytometry, we quantitatively evaluated anti-tumor-promoting activity of rice bran extracts by measuring inhibition of Epstein-Barr virus early-antigen activation (EBV-EA) induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This assay measures anti-tumor-promoting activity and cytotoxicity of target substances using the same batch of cells. Using this short-term procedure, we have determined the anti-tumor-promoting activity of 70% ethanol-water extracts of bran (outer layer) from seeds of five pigmented rice cultivars: Jumlalocal-1, DZ 78, Elwee, LK1-3-6-12-1-1, and LK1A-2-12-1-1. The results show that, compared to an extract from the non-pigmented white cooking rice variety Chuchung, the extracts from the pigmented varieties strongly inhibited phorbol ester-induced tumor promotion in marmoset lymphoblastoid cells B95-8 in vitro. These findings may facilitate development and use of new health-promoting rice varieties.

A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II.

The dentin sialophosphoprotein (DSPP) gene (4q21.3) encodes two major noncollagenous dentin matrix proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Defects in the human gene encoding DSPP cause inherited dentin defects, and these defects can be associated with bilateral progressive high-frequency sensorineural hearing loss. Clinically, five different patterns of inherited dentin defects are distinguished and are classified as dentinogenesis imperfecta (DGI) types I, II, and III, and dentin dysplasia types I and II. The genetic basis for this clinical heterogeneity is unknown. Among the 11 members recruited from the studied kindred, five were affected with autosomal dominant DGI type II. The mutation (g.1188C-->G, IVS2-3C-->G) lay in the third from the last nucleotide of intron 2 and changed its sequence from CAG to GAG. The mutation was correlated with the affection status and was absent in 104 unaffected individuals (208 alleles) with the same ethnic and geological background. The proband was in the primary dentition stage and presented with multiple pulp exposures. The occlusal surface of his dental enamel was generally abraded, and the dentin was heavily worn and uniformly shaded brown. The dental pulp chambers appeared originally to be within normal limits without any sign of obliteration, but over time (by age 4), the pulp chambers became partially or completely obliterated. The oldest affected member (age 59) showed mild hearing loss at high-frequency (8 kHz). Permanent dentition was severely affected in the adults, who had advanced dental attrition, premature loss of teeth, and extensive dental reconstruction.

Differential expression patterns of Runx2 isoforms in cranial suture morphogenesis.

Runx2 (previously known as Cbfal/Pebp2alphaA/AML3), a key transcription factor in osteoblast differentiation, has at least two different isoforms using alternative promoters, which suggests that the isoforms might be expressed differentially. Haploinsufficiency of the Runx2 gene is associated with cleidocranial dysplasia (CCD), the main phenotype of which is inadequate development of calvaria. In spite of the biological relevance, Runx2 gene expression patterns in developing calvaria has not been explored previously, and toward this aim we developed three probes: pRunx2, which comprises the common coding sequence of Runx2 and hybridizes with all isoforms; pPebp2alphaA, which specifically hybridizes with the isoform transcribed with the proximal promoter; and pOsf2, which hybridizes with the isoform transcribed with the distal promoter. These probes were hybridized with tissue sections of mouse calvaria taken at various time points in development. Runx2 expression was localized to the critical area of cranial suture closure, being found in parietal bones, osteogenic fronts, and sutural mesenchyme. Pebp2alphaA and Osf2 showed tissue-specific expression patterns. The sites of Pebp2alphaA expression were almost identical to that of pRunx2 hybridization but expression was most intense in the sutural mesenchyme, where undifferentiated mesenchymal cells reside. The Osf2 isoform was strongly expressed in the osteogenic fronts, as well as in developing parietal bones, where osteopontin (OP) and osteocalcin (OC) also were expressed. However, in contrast to Pebp2alphaA, Osf2 expression did not occur in sutural mesenchyme. Pebp2alphaA also was expressed prominently in primordial cartilage that is found under the sutural mesenchyme and is not destined to be mineralized. Thus, Osf2 isoforms contribute to events later in osteoblast differentiation whereas the Pebp2alphaA isoform participates in a wide variety of cellular activities ranging from early stages of osteoblast differentiation to the final differentiation of osteoblasts.

Clinical Features and Prognosis of Lung Cancer with Brain Metastasis.

PURPOSE: Brain metastasis is estimated to occur in 20~40% of solid tumor patients and the most common primary tumor is lung cancer. Even though the prognosis of brain metastasis is grave and the 1-year survival rate is only 15%, symptom palliations are made with whole brain radiation therapy. We retrospectively evaluated the clinical features and prognostic factors of lung cancer with brain metastasis. MATERIALS AND METHODS: From January 1987 to October 1999, 50 lung cancer patients with brain metastasis underwent whole brain radiation therapy. We reviewed the improvement in neurologic symptoms and survival according to the following parameters; performance status, histological type, presence of brain metastasis at the initial diagnosis of lung cancer, presence of extracranial metastasis, multiplicity of brain lesion, presence of primary lung symptom and treatment modalities. RESULTS: The most frequent symptom with brain metastasis was a headache (50%). Palliation of the headache and other symptoms was achieved in 81% of the patients. Median overall survival after brain metastasis was 21 weeks and the 1 year survival rate was 15%. Patients without extracranial metastasis had a longer median survival than those with, 38 weeks versus 15 weeks, respectively (p=0.01). CONCLUSION: In lung cancer with brain metastasis, neurologic symptoms can be palliated with whole brain radiation therapy, and in this study among such patients, absence of extracranial metastasis can be a good prognostic factor.

Valgus ankle secondary to acquired fibular pseudoarthrosis in children. Long-term results of the Langenskiöld operation.

Nine cases of acquired absence of the fibular shaft were studied to determine the growth contribution of the distal fibula; in 6 cases the absence was caused by osteomyelitis and in 3 cases by trauma. The average valgus and external rotational deformities were 15.2 degrees and 10 degrees, respectively. In 3 of 7 cases surgically treated with Langenskiöld operation or supramalleolar corrective osteotomy, the valgus deformity recurred during the postoperative growth period. The speculated causes of gradual valgus deformity are the loss of physiologic thrust from the proximal to distal fibula, the tethering effect of contracted soft tissue on distal fibula and early physeal closure of the lateral part of the distal tibia due to continuous, uneven axial overloading. The Langenskiöld operation was found effective for the stability of ankle joint in the initial period, but could not prevent the postoperative revascularization of the ankle. However, it is strongly recommended that any types of prophylactic surgery should be carried out before the development of an epiphyseal deformity of distal tibia, and to prevent secondary osteoarthritis of the ankle joint.

Markers of bone and cementum formation accumulate in tissues regenerated in periodontal defects treated with expanded polytetrafluoroethylene membranes.

Guided tissue regeneration (GTR) is a concept that evolved from the development of membrane-barrier techniques, which allow the repopulation of periodontal wounds by specific cells, resulting in a new attachment apparatus. To help understand the biological mechanisms involved in membrane barrier-led periodontal healing, the present study investigated the macromolecules phenotypic of bone and cementum formation in tissues grown under the GTR barrier by immunolocalization. Periodontal regeneration was initiated by placing barriers on experimentally induced periodontal defects in a Rhesus monkey model. Samples were harvested 6 wk after healing and sections of soft tissues grown under GTR barriers (membrane tissue) were stained with antibodies to bone morphogenetic proteins-2 and 4 (BMP-2, BMP-4), bone morphogenetic protein-7 (OP-1), cementum attachment protein (CAP), osteonectin (OTN) and bone sialoprotein (BSP). Tissues grown in the absence of any barrier device served as a control (control tissue). Membrane periodontal tissues from beneath the ePTFE membrane were comprised of spindle-shaped fibroblast-like cells encased in a dense fibrillar extracellular matrix (ECM). Round-shaped cells aggregated to form nodules. Newly formed hard tissue was conspicuous. A similar, but very disorganized, fiber network was observed in control tissues, but neither nodule formation nor hard tissue was observed. Osteonectin staining was observed in the ECM of membrane tissues and particularly in the area of the connective tissue adjacent to newly formed hard tissue. The dense network of connective tissue fibers was also stained. In control tissues, cells and fiber network had a significantly weaker signal for osteonectin. An intense reaction was observed in membrane tissues stained for BSP, particularly the connective tissue adjacent to the newly formed hard tissue, while the control tissues did not stain for BSP. Cementum attachment protein (CAP) was observed in the connective tissue adjacent to the newly formed hard tissue of the membrane tissues whereas control tissues exhibited no CAP staining. In membrane tissues, BMP-2 and 4 distribution was found to concentrate in nodule areas, in the newly formed hard tissue and in the fiber network, while very faint staining was observed in control sections. The distribution of OP-1 in membrane and control tissues was found to mimic the BMP-2 pattern, but staining was more distributed in hard tissue matrix. When the profile of BMP-2, BMP-4, OP-1, OTN, CAP and BSP staining was analyzed on membrane tissue sections, striking similarities were noted in the connective tissue adjacent to the newly formed hard tissue and in nodular areas. In addition, the localization of BMP-2 and BMP-4 mRNA was investigated in both tissues by in situ hybridization. An intense expression of BMP-2 and 4 transcripts was observed in membrane tissues while control tissues never yielded any positive hybridization signal. The correlation between these histochemical findings strongly suggests that the forming soft tissues under ePTFE membranes contain cells and ECM macromolecules normally associated with bone and cementum.

Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I.

For study of the pol gene expression of human T-cell leukemia virus type I (HTLV-I), RNA was transcribed in vitro from proviral DNA and translated in rabbit reticulocyte lysates. This cell-free translation resulted in two major translation products representing the Gag and Gag-Pro polyproteins. By contrast, the Gag-Pro-Pol polyprotein could be readily observed only when translation was performed with mutant mRNA in which the protease (pro) reading frame was aligned to gag to eliminate the frameshifting event in the gag-pro overlap. The results indicated that two independent ribosomal frameshifting events are required for expression of the HTLV-I pol gene product. Studies with mutant DNAs facilitated the characterization of the primary structure of the HTLV-I mRNA responsible for the ribosomal frameshift in the pro-pol overlap and demonstrated that the frameshift occurs at the signal sequence UUUAAAC. Direct amino acid sequencing of the transframe protein localized the site of the frameshift to the asparagine codon AAC.

Identification of HTLV-I gag protease and its sequential processing of the gag gene product.

The full-length provirus of human T-cell leukemia virus type I (HTLV-I) was isolated from MT-2, a lymphoid cell line producing HTLV-I. In transfected cells, structural proteins of HTLV-I, the gag and env products, were formed and processed in the same manner as observed in MT-2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open-reading frame. The deduced amino acid sequences show that this open-reading frame encodes a putative HTLV-I protease. The protease gene (pro) of HTLV-I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag- and protease-coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans-action. A change in the sequence Asp(64)-Thr-Gly, the catalytic core sequence among aspartyl proteases, to Gly-Thr-Gly was shown to abolish correct processing, suggesting that HTLV-I protease may belong to the aspartyl protease group. The 76k gag-pro precursor polyprotein was identified, implying that a cis-acting function of HTLV-I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans-action of the released protease to form p19, p24, and p15.

Pulse oximetry during shunt surgery in pediatric patients with tetralogy of fallot.

Pulse oximetry is a noninvasive technique for measuring O2 saturation (SpO2) continuously. We applied pulse oximetry to 9 pediatric patients with tetralogy of Fallot during shunt surgery. Arterial oxygen tensions (PaO2) and saturations (SaO2) were also measured at the time of postinduction, just before the shunt, after the shunt and at the end of the operation. The SpO2 and SaO2 levels were identically changed in all 4 periods. The PaO2 was increased a little without statistical significance after the shunt procedure and at the end of the operation compared with the values before the shunt. However, SaO2 values increased with statistical significance after the shunt procedure and SpO2 values also showed similar increases with significance. In conclusion, continuous monitoring of SpO2 by pulse oximetry, instead of PaO2, is a very useful and reliable method to assess the improvement of perfusion after shunt, particularly in cyanotic cases.

Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing.

The posttranscriptional regulator (p27x-III) of human T cell leukemia virus type I (HTLV-I) is located predominantly in the cell nucleolus. A highly basic amino-terminal sequence (NH2-Met-Pro-Lys-Thr-Arg-Arg-Arg-Pro-Arg-Arg-Ser-Gln-Arg-Lys-Arg-Pro-Pro -Thr- Pro) in this protein, when fused to the amino termini of beta-galactosidase and p40x of HTLV-I, acts as an autonomous signal capable of directing the hybrid proteins to the cell nucleolus.

Processing of gag precursor polyprotein of human T-cell leukemia virus type I by virus-encoded protease.

The biological activity encoded in the putative protease gene (pro) of human T-cell leukemia virus type I was investigated by using a vaccinia virus expression vector. The 53-kilodalton gag precursor polyprotein was processed into the mature p19, p24, and p15 gag proteins when the gag and protease-coding sequence was expressed under the control of a vaccinia virus promoter, suggesting that the protease may be synthesized through the mechanism of ribosomal frame shifting. The processing defect of a protease mutant could be complemented by cointroduction of a wild-type construct into the cell, demonstrating that the pro gene encodes the biologically active protease molecules which are capable of processing the gag precursor polyprotein in vivo in trans. A study involving the use of a variety of mutants constructed in vitro revealed that the protease consists of a nonessential carboxy-terminal region and a part essential for its activity, including the putative catalytic residue, aspartic acid. Furthermore, a cluster of adenine residues positioned at the overlapping region between the gag and pro genes was shown to be involved in the ribosomal frameshifting event for the synthesis of protease. To mimic the formation of the 76-kilodalton gag-pro precursor polyprotein formed by ribosomal slipping, the coding frames of the gag and pro gene were adjusted. The processing of the gag-pro precursor polyprotein depended on an intact protease gene, implying that a cis-acting function of human T-cell leukemia virus type I protease may be necessary to trigger the initial cleavage event that leads to the release of protease from the precursor protein.

Identification of a protease gene of human T-cell leukemia virus type I (HTLV-I) and its structural comparison.

We determined the nucleotide sequence of a region between the gag and pol genes of a replication-competent proviral clone of a human T-cell leukemia virus type I (HTLV-I) from MT-2 cells. This region overlapping the gag and pol genes contains an open reading frame with a different phase from others. The deduced amino acid sequences show significant homology with the known protease gene of other retroviruses, and harbors highly conserved amino acid sequences that are well conserved in other retroviral protease domains. These results indicate that this open reading frame encodes a HTLV-I protease.

Structural analysis of p28 adult T-cell leukaemia-associated antigen.

The 28,000 mol. wt. polypeptide (p28) of adult T-cell leukaemia-associated antigen encoded by the 24S defective human T-cell leukaemia virus (HTLV-I) is associated with protein kinase activity. We have determined the nucleotide sequence of this defective HTLV-I provirus and found that it contains a portion of the gag gene (p19 and part of p24), the pX region, and two long terminal repeats, one at each end. The predicted p28 gag-pX fused protein consists of 190 amino acids and its mol. wt. was calculated as 21,055. The results of peptide mapping analysis showing that p28 contains p19 supported the nucleotide sequence data. That p28 was encoded by this defective provirus was also demonstrated by transient expression of p28 polypeptide in COS 7 cells transfected with a recombinant plasmid containing a simian virus 40 early promoter and the p28-coding region of the 24S HTLV-I.

Molecular cloning of cDNA encoding gp68 of adult T-cell leukaemia-associated antigen: evidence for expression of the pX IV region of human T-cell leukaemia virus.

A 2.3 kb cDNA was cloned from human T-cell leukaemia virus [HTLV(MT-2)] virion RNA using a vector system, as plasmid pHTLV 707. The restriction endonuclease map of pHTLV 707 revealed that the insert contained the 5' half of the env gene and a portion of the pX region of HTLV, corresponding to the subgenomic RNA derived from 32S defective HTLV. Nucleotide sequence analysis of pHTLV 707 indicated that the clone contained an open reading frame for a 60K mol. wt. protein including the upstream and entire pX IV region. A rabbit antibody raised against a synthetic decapeptide deduced from the nucleotide sequence at the carboxyl terminus of the pX IV region immunoprecipitated gp68, and also 80K and 40K proteins.